PEPTIDOGLYCAN BIOSYNTHESIS AND VANCOMYCIN RESISTANCE

这项建议侧重于细菌细胞壁组装中的酶 肽聚糖(PG)是细菌特有的一种结构，已知 成为几种临床上有用的抗生素的靶标。实验 建议在两个方面：(1)PG中的第一个承诺步骤 生物合成--从PEP到UDP-N-乙酰的不寻常的烯醇式丙酮基转移 生产支架元件UDPenolopruvyl G1cNAc的葡萄糖-Samine 和(2)PG的D-ALA-D-ALA末端形成 抗生素万古霉素的高亲和力部位。我们最近做了 克隆、测序和纯化得到均一的烯醇式丙酮基MurZ 并建议研究其催化机理和催化活性。 抗生素对该酶的时间依赖性失活机制 磷霉素，一种欧洲临床使用的环氧丙烷膦酸盐。不是 关于磷霉素的特异性的分子信息是已知的 MurZ和对催化机理的结构/功能研究可能导致 针对这一目标改进了抗生素设计。 危及生命的革兰氏阳性细菌对万古霉素产生耐药性 凡耐药基因编码五种新基因时的感染(如心内膜炎) 蛋白质，货车，R，H，A，X。我们最近生产过剩，提纯和 研究了D-酮酸还原酶VanH和D-丙氨酸还原酶Vana的特性. D-X连接酶，并表明它们协同作用生成D-丙氨酸-D-乳酸(a 并允许替换正常的D-丙氨酸-D-丙氨酸PG末端 D-丙氨酸-D-乳酸，不再识别万古霉素。我们建议 为进一步研究万古霉素耐药的分子机制 VANA与染色体D-ALA-D-ALA连接酶的比较 提纯和表征VANS和VanR，一种建议的两个组分 调控VANH、A、X转录的调控系统。了解以下内容 VAN耐药蛋白可能允许药物的设计，如PHO- 苯丙氨酸二肽类药物，使万古霉素耐药细菌恢复到 敏感度。

This proposal focuses on enzymes in bacterial cell wall assembly of the peptidoglycan (PG) component, a structure unique to bacteria and known to be the target of several clinically useful antibiotics. Experiments are proposed in two areas: (1) the first committed step in the PG biosynthesis, an unusual enolpyruvyl transfer from PEP to UDP-Nacetyl gluco-samine to produce UDPenolpyruvyl G1cNAc, the scaffolding element for peptide assembly and (2) the D-ala-D-ala termini of PG that form the high affinity site for the antibiotic vancomycin. We have recently cloned, sequenced and purified to homogeneity MurZ, the enolpyruvyl transferase, and propose to study its catalytic mechanism and the mechanism of time-dependent inactivation of this enzyme by the antibiotic fosfomycin, an epoxypropane phosphonate in clinical use in Europe. No molecular information is known about the specificity of fosfomycin for MurZ and structure/function studies on catalytic mechanism could lead to improved antibiotic design against this target. Vancomycin resistance arises in life-threatening gram positive bacterial infections (e.g., endocarditis) when Van resistance genes encode five new proteins, VanS, R, H, A, X. We have recently overproduced, purified and characterized VanH, a D-specific a-ketoacid reductase and VanA, a D-Ala- D-X ligase and shown that they act in concert to make D-Ala-D-Lactate (a depsipeptide) and allow replacement of the normal D-Ala-D-Ala PG terminus by D-Ala-D-Lactate and that no longer recognizes vancomycin. We propose to further study the molecular mechanism of vancomycin resistance by comparison of VanA with the chromosomal D-Ala-D-Ala ligases as well as to purify and characterize VanS and VanR, a proposed two component regulatory system for control of VanH, A, X transcription. Knowledge of the Van resistance proteins may permit design of drugs, such as phos- phinate dipeptidomimetics, to revert vancomycin resistant bacteria to sensitivity.